Maintenance of water and solute homeostasis is critical to survival of the erythrocyte. Primary disorders of erythrocyte hydration are a group of inherited disorders ranging from dehydrated to overhydrated cells. Depending on the degree of perturbation of volume homeostasis, hemolytic anemia may result. Secondary disorders of erythrocyte hydration occur when perturbation in cell hydration is associated with another condition, for instance the dehydration that commonly accompanies sickle cell disease or beta hemoglobinopathies. In secondary disorders, altered erythrocyte hydration may be a major contributor to disease pathology. PIEZO1 has recently been identified as the long sought after protein involved in mammalian mechanosensation and stretch-activated cation channel activation. We have discovered mutations in PIEZO1 that lead to hereditary xerocytosis (HX), a hemolytic anemia characterized by primary erythrocyte dehydration, indicating PIEZO1 plays an important role in cellular volume homeostasis. PIEZO1 is a candidate for unidentified stretch-induced cation pathways in the erythrocyte that play critical roles in erythrocyte aging, malaria invasion, and circulatory sheer stress. PIEZO1 is also an excellent candidate for Psickle, an unidentified cation permeability pathway in sickle erythrocytes at the initiation of the dehydratio cascade of fundamental importance to sickle cell pathobiology. Despite its importance, we have no knowledge of the mechanisms controlling PIEZO1 expression, regulation, structure or function and its role in regulation of volume homeostasis in erythroid cells. The proposed studies combine state of the art cellular, genetic, proteomic, and physiologic technologies to characterize the expression, structure and function of PIEZO1, in an innovative, multidisciplinary manner. Studies include functional, cell-based assays of PIEZO1 membrane protein expression, trafficking, and electrophysiology in a novel, in vivo stably-transfected, single-copy, inducible cll model of PIEZO1 expression. New genetically modified murine models of Piezo1, including a murine model of HX, will be created and characterized. Finally, quantitative MRM-based proteomic studies and state-of-the-art mechanotransduction physiologic techniques will be applied to erythrocytes from HX patients under a variety of cellular conditions. PIEZO1 is found in many cell types including lymphocytes, endothelial, kidney, and neural cells, indicating it likey mediates important functions in a wide variety of cells. Thus studies in erythroid cells may yield mechanistic or biological principles generalizable to many critical cellular processes or human diseases.